Production of renewable fuel from lignocellulosic plant biomass is based on extraction of sugars from plant cell wall material. This extraction process is hampered by the presence of lignin in the cell wall. Lignins contribute to plant “recalcitrance”, a term referring to the inherent resistance of plant material to release polysaccharides and other desirable biomaterials from an interwoven matrix of desirable and undesirable materials (Lynd L R. et al., 1991, Science 251:1318-1323). Lignins are difficult to break down by physical, chemical and other methods, and processing plant materials to release sugars from lignins requires extensive thermochemical or enzymatic treatment. In addition, lignin processing creates inhibitory byproducts, such as acetylated compounds, that hamper further extraction and fermentation. Acetyl esters released during treatment of cell wall polymers can inhibit saccharification of biomass. The released acetate is also inhibitory to the organisms used to ferment the sugars into useful byproducts. Overcoming plant recalcitrance to releasing biomaterials bound in the cell wall is therefore an issue of primary importance in the development of biofuel technology. Finding ways to alter cell wall composition or structure and reduce the severity of pretreatments is a key goal in developing cost-effective biomass feedstocks for biofuel and bioproduct production. The ability to genetically modify biomass feedstocks can have a direct impact on the ability to extract sugars and therefore yield of transportation fuels from plant biomass. Identifying genes that regulate cell wall biosynthesis and composition and reduce recalcitrance is a critical step for efficient production of biofuel and bioproducts from lignocellulosic biomass.
Lignins, complex interlinking biopolymers derived from hydroxyphenylpropanoids, provide rigidity and structure to plant cell walls for plant growth and transport of water and nutrients, and are significant contributors to plant recalcitrance. Lignins are composed primarily of syringyl (S), guaiacyl (G) and p-hydroxyphenyl (hydroxyl-coumaryl) (H) monolignol subunits, which are derived from sinapyl, coniferyl and p-coumaryl alcohols, respectively. The S/G subunit ratio and resulting structure of plant lignins varies according to the genotype, environment, tissue type and maturity of the plant and as such, lignins are very heterogeneous and can vary significantly between different plants, within different tissues of a single plant and even within a single plant cell (Simmons B A et al., 2010, Curr Opin Plant Biol. 13:313-20). This complexity and heterogeneity hinders the development of conversion technology able to process a range of sustainable feedstocks in a cost-effective manner.
Modifying or regulating linkages of lignin with phenolics has been shown to greatly affect biomass digestibility (Li et al., 2014, PLoS One, 9, e105115; Wilkerson et al., 2014, Science, 344, 90-93). On the other hand, high-level lignin has been shown to be a positive factor on biomass saccharification in rice mutants (Li et al., 2015, Plant Biotechnol. J. 13, 514-525; Wu et al., 2013, Biofuels, 6, 183) and artificial cellulose-lignin interactions affect digestibility (Zhang et al., 2016, Bioresour. Technol. 200, 761-769), indicating the level of complexity of cell wall interactions and mechanisms. Properties of the cell wall, including composition, intermolecular interactions and interlinking, cellulose crystallinity and even the release of toxic compounds during pretreatment are all factors that affect accessibility and utilization of sugars for biofuel production.
The genus Populus represents an economically important tree crop that has been targeted for use in diverse applications from the pulp and paper industry, carbon sequestration and as a feedstock in the lignocellulosic biofuel industry (Dinus R J. et al., 2001, Crit. Rev. Plant Sci. 20:51-69).
Identification and manipulation of genes regulating cell wall biosynthesis and recalcitrance is critical both for efficient production of cellulosic sugars and biofuels from plant biomass, and for production of improved cellulose-based products, such as paper and pulp.
Laccases are copper-containing glycoproteins found in a wide range of organisms (Baldrian, 2006, FEMS Microbiol. Rev. 30, 215-242; Claus, 2003, Arch. Microbiol. 179, 145-150; Dittmer and Kanost, 2010, Insect Biochem. Mol. Biol. 40, 179-188; Dittmer et al., 2004, Insect Biochem. Mol. Biol. 34, 29-41; McCaig et al., 2005, Planta, 221, 619-636.). Although they share significant homology, laccases appear to have functionally diverged within and between phylogenetic clades (Dittmer et al., 2004, Insect Biochem. Mol. Biol. 34, 29-41). Bacterial, fungal and insect laccases have been shown to function in the degradation of lignin, whereas higher plant laccases are thought to function in the polymerization of lignin subunits (Sharma and Kuhad, 2008, Indian J. Microbiol. 48, 309-316). Additionally, even though laccases retained similar protein domains, molecular modelling suggests differences in protein folding and affinity for interacting with lignin, which may result in divergence of activity in lignin synthesis and degradation (Awasthi et al., 2015, J. Biomol. Struct. Dyn. 33, 1835-1849). Laccases are known to function in oxidation reactions involving various inorganic and organic substrates including phenolics and aromatic amines in plants. Studies in Populus and Arabidopsis suggest that laccases act not only in the biosynthesis of lignin but also may contribute to additional roles of cell wall chemistry or integrity (Ranocha et al., 2002, Plant Physiol. 129, 145-155; Ranocha et al., 1999, Zhao et al., 2013). In plants, it was thought that laccases may be involved in lignin biosynthesis based on their capability to oxidize lignin precursors and their localization in lignifying tissues (Bao et al., 1993, Driouich et al., 1992; Ranocha et al., 1999, Eur. J. Biochem. 259, 485-495; Sterjiades et al., 1992, Plant Physiol. 99, 1162-1168). For example, over-expression of the cotton laccase, GaLACCASE 1 (LAC1), in Populus leads to increased lignin content with transgenic plants showing a 2.1%-19.6% increase in total lignin, indicating that laccases are involved in lignin biosynthesis (Wang et al., 2008, Plant Cell Tissue Organ Cult. 93, 303-310). In Arabidopsis, insertional mutations in three laccase-encoding genes completely abolished lignin accumulation (Zhao et al., 2013, Plant Cell, 25, 3976-3987). Interestingly, the three laccases, AtLAC4, 11 and 17, are not paralogous and show homology to different subfamilies of the laccase gene family, suggesting that lignin biosynthesis is not controlled by a single subfamily. A study in Populus indicated that transgenic trees, in which expression of the laccase gene PtLAC3 was reduced, showed a threefold increase in phenolic content which accumulated in xylem ray parenchyma cells (Ranocha et al., 2002, Plant Physiol. 129, 145-155). In addition, xylem fibre cell walls were dramatically altered leading to severe deformation, indicating a defect in cell wall integrity and supporting the importance of this laccase in normal xylem cell wall structure and integrity. However, there was no significant change in lignin quantity or composition. (Ranocha et al., 2002, Plant Physiol. 129, 145-155).